Laboratory-selected colonies of western corn rootworm with increased tolerance to maize containing event das-59122-7

ABSTRACT

Laboratory-selected colonies of western corn rootworm exhibiting tolerance to maize containing event DAS-59122-7 are described. Further, methods for various uses of these resistance western corn rootworm colonies are also described, including development of negative cross-resistance strategies and improved resistance management strategies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to provisionalapplications Ser. No. 60/977,477 filed Oct. 4, 2007, and Ser. No.61/029,958 filed Feb. 20, 2008, herein incorporated by reference intheir entirety.

BACKGROUND OF THE INVENTION

The present invention relates to new laboratory-selected colonies ofwestern corn rootworm (WCR, Diabrotica virgifera virgifera, LeConte)with increased tolerance to maize containing event DAS-59122-7, as wellas methods of using such tolerant organisms and the information gatheredfrom such organisms.

Maize (zea mays) is often referred to as corn in the United States. Onemajor problem faced by growers of maize in the United States is theeffects of pests, such as WCR, on the yield and standability of aparticular maize crop. In an effort to combat pest infestations, variousmethods have been employed in order to reduce or eliminate pests in aparticular plot. These efforts include rotating corn with other cropsthat are not a host for a particular pest, applying pesticides to theabove-ground portion of the crop, applying pesticides to the soil in andaround the root systems of the affected crop, and utilizing maize plantsincorporating transgenic genes which cause the maize plant to produceinsecticidal proteins providing protection from the target pest(s).

A recurring problem with these types of pest management strategies isthe development of resistance in the pest population. If a particularpest management strategy is used for a long enough period of time,eventually pests that are resistant to the particular pesticidalstrategy utilized will be selected for, and the pest population willeventually be predominantly comprised of pests with resistance to thepest management strategy. Once this occurs, the strategy or particularinsecticidal tactic previously used is no longer effective, and effortsmust be undertaken to determine a new method to reduce or eliminate thetarget pest.

Although efforts have been made to slow the development of resistance topesticides, the evolution of resistance is generally consideredinevitable when the pest management tactic applies some selectivepressure. Once widespread resistance develops, the chemical (orchemical-class) that resistance has developed against is typicallyabandoned. The subsequent focus in the research and industrial communityis to identify novel pesticides and antibiotics with different modes ofaction, where positive cross-resistance to previously used toxicantsdoes not occur.

The most commonly-used strategy to manage or slow the development ofresistant pests in crops is by use of a refuge. A refuge is a source forsusceptible pests to survive on untreated or non-pest resistant sources.In this regard, the refuge permits susceptible pests to survive and growto adulthood, allowing them to mate with pests exhibiting tolerance orresistance to the pest management strategy, thereby diluting theprevalence of the gene(s) conferring resistance or tolerance. Undercurrently-accepted guidelines, a minimum of 20% corn refuge is typicallyused in order that sufficient susceptible pests survive to adulthood. Ofcourse, this method has the obvious drawback of leaving 20% of a corncrop susceptible to pest attack, thereby reducing yield substantially inthose plants.

Another alternative to discarding old compounds and continually seekingnew compounds is the development of negative cross-resistance strategiesto control organisms containing the resistance allele. Negativecross-resistance (NCR) as a strategy for insecticide resistancemanagement refers to a scenario where organisms tolerant to one compoundare highly sensitive to another compound and vice versa. For example, ifone treats an insect population with a toxin such as pesticide “A,” thenumber of insects carrying alleles resistant to pesticide “A” willincrease in frequency. After numerous generations, insects carrying the“A” resistance allele will comprise the majority of the population. Atthis time a second toxin that preferentially kills those insectstolerant to the first toxin is used to treat the insect population. Useof the second toxin changes the frequency of the alleles such that thefirst toxin can again be used to control the insects' population for oneto several generations. By alternately deploying the two toxins, a NCRstrategy can be used to maintain effective control of the pest while‘managing’ the resistance alleles in the insect population.

In order to develop negative cross resistance strategies, however, it isnecessary to be able to comparatively test susceptible and tolerantorganisms to determine compounds which have a higher efficacy againstresistant or tolerant organisms as compared to susceptible organisms.This problem is complicated by the manner by which resistant or tolerantpests are typically obtained, namely by direct and potentiallyunrealistic exposure to the toxin of interest. This type of exposuredoes not accurately reflect the conditions under which pests willencounter toxins in the field, however, such as in the case of toxinsproduced by transgenic pest resistant crops.

In addition, when resistant or tolerant organisms are available, theconverse effect may be identified, namely positive cross-resistance. Inthis instance, organisms that are resistant or tolerant to a given toxinalso show increased resistance or tolerance to another, different toxin.When this phenomenon is observed, there is a greater danger ofresistance or tolerance development in the target pest, as developmentof resistance or tolerance to one toxin also increases resistance ortolerance to another toxin.

Resistant and tolerant organisms are also beneficial for genetic study.An understanding of the genetics that confer tolerance or resistance toa toxin can be highly beneficial in many respects, including designingnew toxins or new versions of existing toxins, understanding themechanism of resistance development, assisting in determining howresistance may be delayed from a genetic or other perspective anddetermining how resistance may be delayed if there are multiple andindependent traits in the same target pest that confer different levelsof resistance.

BRIEF SUMMARY OF THE INVENTION

Western corn rootworm colonies were selected from approximately 2,000wild western corn rootworm insects collected from the field. Theinventors believe they are the first to identify and selectivelypropagate colonies of western corn rootworm which are tolerant totransgenic maize event DAS-59122-7. The colonies were developed based oncollections made near York, Nebr. and Rochelle, Ill. They have thus beendesignated the “York Selected Colony” and the “Rochelle SelectedColony,” based on the location of the original beetle collection.

This invention thus relates to western corn rootworms that exhibitincreased tolerance to event DAS-59122-7. The invention further relatesto beetles showing increased tolerance to event DAS-59122-7, to eggsshowing increased tolerance to event DAS-59122-7, and to larvae showingincreased tolerance to event DAS-59122-7. The invention further relatesto the York and Rochelle Selected Colonies. This invention also relatesto the descendants of all progeny resulting from mating these organismswith other colonies of western corn rootworm including derivatives ofsubsequent generations identifiable by their tolerance to maizecontaining event DAS-59122-7. This invention further relates to any useof these organisms, including the use and development of a negativecross-resistance strategy, resistance monitoring strategies, refugedeployment strategies, positive cross-resistance determinations, ordetermination of mechanisms of resistance, or any other use.

The invention also relates to use of such organisms in the context of anegative cross-resistance development strategy. The invention alsoincludes use of such organisms to screen for positive cross-resistance.The invention further includes use of such organisms to determine thegenetic basis for tolerance to event DAS-59122-7. The invention furtherrelates to utilization of the organisms to develop novel resistancemanagement strategies. The invention further relates to the use of suchorganisms to validate resistance management assumptions based oncomputer models for resistance-risk for event DAS-59122-7.

Thus, in one aspect, the invention includes a western corn rootworm inany stage that exhibits increased tolerance to event DAS-59122-7, andadditionally to insects in any stage of the York Selected Colony orRochelle Selected Colony.

In another aspect, the invention concerns mating tolerant insects,including those from either or both of the York Selected Colony or theRochelle Selected Colony, with another colony of western corn rootwormto obtain a western corn rootworm that is tolerant to event DAS-59122-7.

In a further aspect, the invention concerns utilizing tolerant insects,including those from either or both of the York Selected Colony orRochelle Selected Colony, to determine potential insect controlstrategies utilizing negative cross-resistance.

In another aspect, the invention concerns utilizing tolerant insects,including those from either or both of the York Selected Colony orRochelle Selected Colony, to develop new resistance managementstrategies.

In another aspect, the invention concerns utilizing tolerant insects,including those from either or both of the York Selected Colony orRochelle Selected Colony, to validate assumptions used in resistancerisk computer simulation models.

In yet a further aspect, the invention concerns utilizing tolerantinsects, including those from either or both of the York Selected Colonyor Rochelle Selected Colony, to evaluate the possibility of positivecross-resistance with other existing or potential commercial cornrootworm control tactics, such as chemical insecticides or othertransgenic maize events.

In other aspects, the invention concerns utilizing tolerant insects,including those from either or both of the York Selected Colony orRochelle Selected Colony, to understand the mechanism of western cornrootworm and other Diabrotica spp. resistance to various insect controlstrategies, such as, for example, Bt toxins.

In another aspect, the invention relates to utilizing tolerant insects,including those from either or both of the York Selected Colony orRochelle Selected Colony, as a reference to monitor populations ofwestern corn rootworms in the field.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows realized heritability (h²) of the survival characteristicover 10 and 11 generations of selection using DAS-59122-7 for Rochelle-Sand York-S colonies, respectively.

FIG. 2 compares observed (columns) mean population fitness of Rochelle-Sand York-S colonies across generations of selection with DAS-59122-7plotted with expected mean population fitness (lines) of the samecolonies assuming they contained alleles for a putative major resistancegene.

FIG. 3 shows population genotypic frequencies over generations using theWCR benchmark simulation excluding the tolerance trait (Y), andexcluding any block or blended refuge.

FIG. 4 shows population genotypic frequencies over generations using theWCR benchmark simulation excluding the tolerance trait (Y) and includinga block refuge at a proportion of 0.20.

FIG. 5 shows population genotypic frequencies over generations using theWCR benchmark simulation excluding the putative resistance gene (B), andexcluding any block or blended refuge.

FIG. 6 shows population genotypic frequencies over generations using theWCR benchmark simulation, and excluding any block or blended refuge.

DETAILED DESCRIPTION

Two separate western corn rootworm colonies that exhibit tolerance tomaize event DAS-59122-7 were selected via laboratory breeding process.The process by which the colonies were developed is described below.

York Selected Colony

1. The York Selected Colony was initiated by collecting approximately6,000 western corn rootworm adults from the field near York, Nebr. inAugust of 2002.

2. Beetles were caged in the laboratory and approximately 30,000 eggswere collected and stored at 10° C. for 5-6 months, and then incubatedat 25° C. until initial hatch was observed. Eggs were then infested ontoseedling maize and reared to adults.

3. A bulk cross was made of approximately 1,000 males from York withapproximately 1,000 virgin females from a non-diapausing colony ofwestern corn rootworm. Introgressing the non-diapausing trait eliminatesobligate diapause and enables more rapid cycling of rootworm populationselection.

4. Eggs produced from this cross were held and allowed to hatch withoutundergoing a 5-6-month obligatory cold period. The result of this crossand selection for egg hatch was a half wild-type non-diapausing colony.This process initiated the York colony.

5. The York colony was first selected in May 2004 with eggs from thefifth generation after introgressing the non-diapausing trait.Approximately 12,000 Rochelle colony eggs were infested onto a seedlingmaize hybrid containing event DAS-59122-7 and insects were reared toadulthood. A total of 117 males and 34 females survived to adulthoodduring this initial generation of continuous exposure to eventDAS-59122-7. This process resulted in the F1 generation of the YorkSelected Colony (Table 1).

6. All F1 survivors were caged together, allowed to mate, and all oftheir 9,707 eggs were infested back onto event DAS-59122-7 maizeseedlings. Two hundred nine males and 86 females survived to adulthoodduring this second round of selection. This process of using DAS-59122-7maize plants to selected for resistance in the York Selected Colony wasrepeated for a total of 11 generations.

On two separate instances, the number of adults recovered from selectionwas sufficiently low that the York Selected Colony was reared throughone generation on conventional corn to provide sufficient adults andsubsequent eggs to continue the selection process. This method ofincreasing the population size was used after the F1 and F10 selections.

Rochelle Selected Colony

1. The Rochelle colony was initiated by collecting approximately 4,000western corn rootworm adults from a field plot of pumpkin (Cucurbitamaxima L.) located near Rochelle, Ill. in August of 2002.

2. Beetles were caged in the laboratory and approximately 30,000 eggswere collected and stored at 10° C. for 5-6 months, and then incubatedat 25° C. until initial hatch was observed. Eggs were then infested ontoseedling maize and reared to adults.

3. A bulk cross was made of approximately 1,000 males from Rochelle withapproximately 1,000 virgin females from a non-diapausing colony ofwestern corn rootworm. Introgressing the non-diapausing trait eliminatesobligate diapause and enables more rapid cycling of rootworm populationselection.

4. Eggs produced from this cross were held and allowed to hatch withoutundergoing a 5-6-month obligatory cold period. The result of this crossand selection for egg hatch was a half wild-type non-diapausing colony.This process initiated the Rochelle colony.

5. The Rochelle colony was first selected in August 2004 with eggs fromthe sixth generation after introgressing the non-diapausing trait.Approximately 18,000 Rochelle colony eggs were infested onto a seedlingmaize hybrid containing event DAS-59122-7 and insects were reared toadulthood. A total of 89 males and 46 females survived to adulthoodduring this initial generation of continuous exposure to eventDAS-59122-7. This process resulted in the F1 generation of the RochelleSelected Colony (Table 1).

6. All F1 survivors were caged together, allowed to mate, and all oftheir 8,592 eggs were infested back onto event DAS-59122-7 maizeseedlings. Fifty five rootworms survived to adulthood during this secondround of selection. This process of using DAS-59122-7 maize plants toselected for resistance in the Rochelle colony was repeated for a totalof 10 generations.

On two separate instances, the number of adults recovered from selectionwas sufficiently low that the Rochelle Selected Colony was rearedthrough one generation on conventional corn to provide sufficient adultsand subsequent eggs to continue the selection process. This method ofincreasing the population size was used after the F1 and F9 selections.

During the colony selection process, various experiments were conductedin order to ascertain the continued efficacy of DAS-59122-7 and thedevelopment of resistance or tolerance to the event. The experimentsconducted are described below.

Mean survival rates were calculated from cohort-specific survival rateswithin a generation. Cohort survival rate was estimated by dividing thetotal number of recovered adults for a cohort by a weighted mean oftotal number of viable eggs exposed to seedlings. The mean number ofviable eggs exposed for each cohort was calculated by first multiplyingthe number of eggs infested per container by the number of containersand by the mean hatch rate for the cohort. The number of viable eggsexposed in a generation was a weighted mean, where each cohort wasweighted based on the number of containers in that cohort relative tothe total number of containers for the entire generation. Meanpercentage female was calculated from gender counts collected across allcohorts. For each colony and generation, adults were pooled into onecage and the number of live females was tracked weekly until the cagewas terminated. Fecundity was estimated by dividing the number of totaleggs per cohort by the number of live females. This estimate wasstandardized across colonies and generations using female and egg valuesfrom the week beginning two weeks after the maximum number of livefemales was recorded. Most often the same cohort of eggs used toestimate fecundity was used to estimate fertility. Otherwise, fertilitywas calculated from the nearest cohort in time. Fertility estimatesresulted from an average within the weekly cohort.

Table 1 reports generational estimates for Rochelle-S and York-S,whereas unselected colony traits were reported as across-generationmeans. Life history trait information was collected from all selectedcolony cohorts across the lifespan of adults, whereas large insectnumbers in unselected colonies limited data collection to one cohort.This difference in the way adult life history trait information wascollected prevented statistical analyses. Table 1 identifies grossdifferences in measured traits among colonies and can be used to improvebiological or genetic parameter estimates used in computer modelssimulating the rootworm and DAS-59122-7 system.

TABLE 1 Rochelle-S York-S Mean Mean Generation Eggs Survival PercentageEggs Survival Percentage Evaluated¹ exposed Rate² Female³ Fecundity⁴Fertility⁵ exposed Rate² Female³ Fecundity⁴ Fertility⁵ F1 8,592 1.3 34.116.8 40.3 9,707 0.4 31.4 11.6 18.0 F2 47,000 2.5 — — — 48,500 8.5 30.718.9 39.0 F3 61,255 7.4 48.2 5.1 45.3 39,863 2.9 34.6 17.8 39.0 F440,110 6.4 43.9 9.6 43.0 13,100 1.6 23.9 26.0 38.6 F5 60,000 15.9 35.510.0 44.0 41,493 8.9 36.3 12.6 42.3 F6 77,000 12.7 36.4 10.3 41.6 53,80015.4 28.4 10.3 51.0 F7 82,000 12.3 29.7 7.4 34.0 79,100 7.0 27.4 12.747.0 F8 94,000 16.7 37.8 12.5 54.0 79,000 11.6 31.7 6.6 31.0 F9 91,00019.7 — — — 94,000 23.4 31.6 9.7 43.0 F10 84,000 13.7 — — — 91,000 17.9 —— — F11 — — — — — 84,000 15.6 — — — Across 10.8 (6.17) 38.0 (6.23) 10.243.2 10.3 (7.28) 30.7 14.0 38.8 (9.6) Generation (3.73) (6.02) (3.74)(5.91) Mean (SD) Rochelle-US York-US Mean Survival Percentage MeanSurvival Percentage Rate² Female³ Fecundity⁴ Fertility⁵ Rate² Female³Fecundity⁴ Fertility⁵ Across 76.2 (29.34) 53.0 (4.40) 18.6 34.5 82.7(45.88) 49.6 25.6 40.1 Generation (9.32) (7.59) (7.08) (6.68) (11.16)Mean (SD) ¹Life history trait measures were collected from the progenysurviving F rounds of selection; for example, F1 measures were collectedfrom rootworm surviving the first round of selection. ²Survival rateestimates reported as the percentage of viable eggs producing adultsusing continuous exposure to either DAS-59122-7 (selected) or isoline(un-selected). ³Number of females are reported as a percentage of thetotal number of adults. ⁴Fecundity estimates are reported as the meannumber of eggs/female/day. ⁵Fertility estimates reported as percentageof eggs hatching in a subsample greater than 100 eggs.

A sublethal seedling assay (SSA) was used to characterize larvalpopulation development for Rochelle-US, Rochelle-S, York-US and York-Scolonies using isoline (susceptible) and DAS-59122-7 maize. The SSA usesrate of larval population development while feeding on maize roots as ameasure of rootworm susceptibility to different types of maize. The SSAprovides ecologically relevant larval exposure to facilitate sensitivemeasures of change in larval population susceptibility.

Samples of eggs from each generation of each colony were evaluated onboth DAS-59122-7 and isoline using the SSA. Three experimental units(containers) of the SSA were made for each colony by maize-typeevaluation. The assay duration was 17 days, after which larvae wereextracted into alcohol using a Berlese funnel. Extracted larvae weredistributed across the bottom of a flat pan marked with a grid pattern.A sub-sample of larvae were collected from each experimental unit byrandomly sampling squares for all larvae within a square until 25 or 30total larvae were collected. Each colony was evaluated on DAS-59122-7and isoline seedlings at the same time using the same rearing andenvironmental conditions. However, different WCR developmental rates onDAS-59122-7 and isoline maize led to running the SSA for differentcolonies on different dates.

Tables 2 and 3 report the colony and generational assays using the SSAtechnique. Table 2 shows the mean percentage of larvae in each instarfor Rochelle-US and Rochelle-S 17 days after egg hatch on DAS-59122-7maize in the sublethal seedling assay test system, and Table 3 shows themean percentage of larvae in each instar for York-US and York-S 17 daysafter egg hatch on DAS-59122-7 maize in the sublethal seedling assaytest system. Only the F1 and F2 generations of York-S were not evaluatedon isoline and are not part of York-S across generation means reportedin Table 3.

TABLE 2 Mean percentage of larvae in each instar¹ Quantile P valueGeneration Rochelle-US Rochelle-S correlation (H0: ρ ≧ evaluated 1^(st)2^(nd) 3^(rd) 1^(st) 2^(nd) 3^(rd) ρ0² (Upper 95% CL)³ ρ0)⁴ F0 9.0 64.027.0 — — — — — — F1 6.7 76.0 17.3 5.3 46.7 48.0 0.78 0.64 (0.73) 0.002F2 6.7 76.0 17.3 0.0 57.3 42.7 0.78 0.55 (0.65) <0.001 F3 10.7 77.3 12.04.0 64.0 32.0 0.71 0.62 (0.71) 0.057 F4 32.0 62.7 5.3 12.0 61.3 26.70.81 0.65 (0.73) <0.001 F5 5.6 75.6 18.9 0.0 22.2 77.8 0.58 0.42 (0.54)0.014 F6 13.3 76.0 10.7 0.0 37.3 62.7 0.74 0.52 (0.63) <0.001 F7 12.076.0 12.0 1.3 54.7 44.0 0.68 0.53 (0.63) 0.007 F8 1.3 76.0 22.7 0.0 25.374.7 0.88 0.34 (0.48) <0.001 F9 17.3 73.3 9.3 1.1 48.9 50.0 0.73 0.54(0.65) <0.001 F10 7.9 89.9 2.2 0.0 61.1 38.9 0.57 0.30 (0.44) <0.001Across generation 11.1 74.8 (7.12) 14.1 (7.39) 2.4 (3.87) 47.9 (15.03)49.8 (17.08) mean (SD) (8.13) ¹Besides F7 un-selected using 50 larvae,all other larval percentage estimates are based on a sub-sample of 75 or90 larvae. ²ρ0 = The largest correlation value used in the Q-Q analysisthat results in no difference (P > 0.05) among within-generationun-selected experimental units. ³Correlation, and upper 95% confidencelimit, between un-selected and selected instar quantiles for eachgeneration. ⁴The quantile analysis tested whether the correlationbetween un-selected and selected strains was equal to or greater thanthe corresponding ρ0.

TABLE 3 Mean percentage of larvae in each instar¹ Quantile P valueGeneration York-US York-S correlation (H0: ρ ≧ Evaluated 1^(st) 2^(nd)3^(rd) 1^(st) 2^(nd) 3^(rd) ρ0² (Upper 95% CL)³ ρ0)⁴ F0 6.0 61.0 33.0 —— — — — — F1 6.7 82.7 10.7 1.3 70.7 28.0 0.79 0.56 (0.66) <0.001 F2 6.782.7 10.7 1.3 68.0 30.7 0.79 0.53 (0.64) <0.001 F3 1.3 56.0 42.7 0.014.7 85.3 0.79 0.39 (0.52) <0.001 F4 — — — — — — — — — F5 8.0 81.3 10.71.3 64.0 34.7 0.84 0.51 (0.62) <0.001 F6 6.7 74.4 18.9 0.0 40.0 60.00.56 0.47 (0.59) <0.001 F7 12.0 81.3 6.7 0.0 38.7 61.3 0.60 0.33 (0.47)<0.001 F8 4.0 85.3 10.7 0.0 42.7 57.3 0.74 0.36 (0.50) <0.001 F9 — — — —— — — — — F10 10.7 81.3 8.0 0.0 26.7 73.3 0.65 0.51 (0.62) 0.019 F11 8.987.8 3.3 0 5.1 48.9 0.70 0.35 (0.48) <0.001 Across generation 7.1 (3.09)77.4 15.5 0.4 (0.65) 41.2 (23.28) 53.3 (19.57) mean (SD) (10.58) (12.61)¹75 or 90 larvae were used to describe the age structure of theun-selected or selected strains for each generation. ²ρ0 = The largestcorrelation value used in the Q-Q analysis that results in no difference(P > 0.05) among within-generation un-selected experimental units.³Correlation, and upper 95% confidence limit, between un-selected andselected instar quantiles for each generation. ⁴The quantile analysistested whether the correlation between un-selected and selected strainswas equal to or greater than the corresponding ρ0.

A total of 6 greenhouse efficacy experiments were conducted periodicallyacross generations to determine if selection changed the injurypotential of Rochelle-S and York-S on DAS-59122-7 roots. The injurypotential of Rochelle-S was evaluated after the F1, F2, F5, and F10generations of selection. The injury potential of York-S was evaluatedafter the F1, F2, F6, and F11 generations of selection. Two unique testsystems were utilized across experiments: a seedling root trainerbioassay and a large pot bioassay. Root trainers were used tocharacterize the injury potential of Rochelle-S F1 and F2 generations.

The experimental unit was a single rootworm-infested maize plant. Theexperimental design consisted of 4 treatments arranged in a randomizedcomplete block design with 4 replications. The experiment was repeatedon 6 consecutive dates each initiated at 7-d intervals. On each date,kernels of both hybrids were planted individually in 3.8×5×20 cm cellsof Rootrainers (Hummert International, Earth City, Mo.) containing asoil-less potting mix formulated for greenhouse use. At growth stage V2,a herbicide leaf-painting technique was performed on each DAS-59122-7seedling to verify presence of the herbicide selectable marker linkedwith DAS-59122-7. Plants with symptoms of herbicide injury (notcontaining DAS-59122-7) were removed from the experiment. Thirteen daysafter planting, each seedling was infested with 100 eggs of theappropriate insect colony pre-incubated to hatch within 7 days ofinfestation. Eggs were infested to each trainer cell using a variablerate pipette calibrated to deliver the desired quantity of eggssuspended in a 0.08% agar solution. Nineteen days after infestation,plants were extracted from the trainers and the root systems visuallyscored for injury using the 0-3 node-injury scale (Oleson et al. 2005).

The large pot test system was used in the remaining 5 greenhouseefficacy experiments. The experimental unit was a singlerootworm-infested maize plant growing in a 7.5 L pot. The experimentaldesign for each pot experiment consisted of 4 treatments arranged in arandomized complete block design with 5 replications. Treatmentsincluded in each pot experiment are shown in Tables 5 and 6. Each potexperiment was initiated by planting kernels of each hybrid into flatsfilled with a soil-less potting mixture. Ten days after planting, theDAS-59122-7 seedlings were leaf painted to verify presence of theselectable marker and plants showing symptoms of herbicide injury werediscarded. Seedlings were then removed from the flats and individuallytransplanted into 7.5 L pots 12-13 d after planting. Infestation datesand the number of eggs infested per seedling are shown in Tables 5 and6. Egg viability for all experiments was determined using the hatch testmethod described previously. Infested plants were maintained in thegreenhouse with regular watering until first beetle emergence wasdetected in the pots. The root systems were then extracted from thepots, washed with pressurized water, and the amount of root injuryscored using the 0-3 node-injury scale (Oleson et al. 2005). Tables 5and 6 report the dates roots were evaluated for each pot experiment.

A non-parametric quantile-quantile (Q-Q) analysis was used to contrastrates of larval population development (Nowatzki et al. submitted,Johnson and Wichern 2002). The Q-Q analysis uses pair-wise comparisonsof the quantiles of unselected and selected strains of WCR by colony. Inthis analysis, the 1st through 100th quantile values (0.01, 0.02 . . .0.99, 1.00) were calculated for each data set. If un-selected andselected colonies are similar in their susceptibility to DAS-59122-7,then the quantiles of the unselected colony should be highly correlatedwith the quantiles of the corresponding selected colony. For eachpopulation and generation tested, an appropriate ρ0 value was determinedusing three replicate data sets from the unselected colony developing onDAS-59122-7. Quantiles were calculated for each replicate and aniterative process was used to identify the largest ρ0 value thatresulted in no significant difference (P>0.05) among the threeexperimental units. This iterative method of estimating an appropriateρ0 from un-selected WCR on DAS-59122-7 incorporates test systemvariation and decreases the Type I error rate. For each generation, thenull hypothesis was that the true correlation between unselected andselected larval samples for either colony is greater than or equal toρ0.

Change in realized heritability (h²), or the proportion of totalphenotypic variation attributable to additive genetic variation, wasestimated from change in the distribution of instars between unselectedand selected colonies over generations for both Rochelle and York usingthe method described by Tabashnik (1992). The distribution of instarsfor each colony at each generation was estimated using a lognormaldistribution. The log_(e) of the median instar and its associatedstandard deviation were estimated from lognormal distribution fits usingthe lognormal option of the Empirical CDF routine in Minitab® Release14.12.0 (Minitab Inc, 2004). Realized heritability (h²) was estimated bycomparing the selected colony (-S) to the susceptible colony (-US) foreach location. The susceptible colonies (-US) served as a reference thatwas subjected to all of the environmental and handling selectionpressures as the selected (-S) colonies. Heritability was calculated foreach generation and location as h²=R/(iS) where R was calculated asR=(log_(e)(median-S)−log_(e)(median-US))/n, “i” was the selectionintensity which was assumed to be 1 since all instars were present ineach sample, and S was the average standard deviation associated withthe instar distributions that was estimated as1/(0.5*(slope-US+slope-S)) that gives the total phenotypic variation ofthe instars. The results were then tabled and plotted over generationsby colony.

Node-injury scores from each greenhouse efficacy experiment wereanalyzed using the general linear model procedure in SAS (SAS 2006) totest for differences in injury potential between treatments. Within eachefficacy experiment, treatment means were compared with a t-test(P=0.05) on the differences of least squares means (PDIFF) in SAS. Thepercentage consistency (percentage of roots with node-injury scores of0.00-0.25, 0.26-0.50, 0.51-0.99, 1.00-1.99, and 2.00-3.00) withintreatments was also calculated. Additionally, mean node-injury scoresadjusted for the number of viable eggs infested (node-injury score per100 viable eggs infested) were also calculated to allow for a relativecomparison of injury potential of the selected colonies acrossgenerations of selection.

Results from selected colony experiments were used to test whether ornot founding Rochelle and York populations may have contained putativeresistance. For the purpose of this assessment, putative or majorresistance is assumed to be conferred by a rare, single, and recessiveallele (r); three possible genotypes may occur at the locus, namely, SS,rS and rr. Only homozygous resistant individuals are assumed to be ableto develop and survive on DAS-59122-7. In this study, the effective sizeof the founder populations was assumed to be sufficiently large toinclude the r allele. The hypothesis, that r was present in the foundingpopulations, was tested by simulating changes in the frequency of ralleles using hypothesis-specific parameters and the standard geneticmodel following Fisher's Fundamental Theorem of Natural Selection(Fisher 1930). Li (1967) provided a mathematical formula for iterativeuse in describing changes in genotypic frequency with selection:

${\Delta \; q} = {\frac{{pq}\left( {{q\left( {{Wrr} - {WrS}} \right)} + {p\left( {{WrS} - {WSS}} \right)}} \right)}{\left( {{p^{2}{WSS}} + {2{pqWrS}} + {q^{2}{Wrr}}} \right)}.}$

where p is the r allele frequency, q is the S allele frequency and Wrr,WrS and WSS are the fitness parameters of rr, rS and SS genotypes,respectively. Li's equation was used iteratively (q(t+1)=q(t)+Δq) tocalculate allele frequencies over t generations (York: t=11, Rochelet=10). Mean population fitness (WM) was estimated for each generationusing the standard equation of Hartl and Clark (1989):

W _(M) =p ² Wrr+2pqWrS+q ² WSS

Simulated mean population fitness was contrasted to observed meanpopulation fitness. Observed mean population fitness on DAS-59122-7 wasthe selected colony survival rate adjusted up assuming the survivalrates of the unselected colony on isoline maize was mean populationfitness equal to 1.0. Normal test system survival rate was estimatedfrom across generation means of the Rochelle-US and York-US populations(Table 1). Contrasts between observed and simulated mean populationfitness across generations of selection were made using a Chi-Squaretest, where the variance term used in the denominator was the sum ofstandard deviations for observed survival rates.

The fitness of these genotypes on DAS-59122-7 was calculated assumingthat 1 in 150 plants in the selection scheme was an off-type or did notcontain DAS-59122-7. Therefore, the fitness values (W) of SS and rSgenotypes are the weighted average of 1/150 fitness on unprotectedplants and 149/150 DAS-59122-7 plants. The mortality rate of asusceptible population of WCR using realistic exposure scenarios wasestimated at 99.75%, or W=0.0025 (Storer et al. 2006). Adjusted for(1/150) off-type seeds, this value increases to 0.0092. Fitness of therS genotype (also dominance (h) of the r allele) was assumed to be 0.08;adjusted for (1/150) off-type seeds this value increases to 0.0862.Assumed fitness of the rr genotype was 1.0. These results areillustrated in FIG. 2.

Table 4 describes the normal developmental rate of all colonies onisoline maize; after 17 days there was less than 1% of larvae still inthe first instar, 4.9 to 18.2% in the second instar and 81.6 to 95.1% inthe third instar. Rochelle-US and Rochelle-S colonies developed atsimilar rates on isoline, and York-US and York-S colonies developed atsimilar rates on isoline. One trend was an increased and less variablerate of larval development for both York colonies compared to bothRochelle colonies.

TABLE 4 Across generation mean (SD) percentage of larvae in each instarColony 1^(st) 2^(nd) 3^(rd) Rochelle-US 0.6 (2.01) 13.2 (21.63) 86.2(23.57) Rochelle-S 0.1 (0.42) 18.2 (16.97) 81.6 (17.17) York-US 0.0(0.00)  4.9 (4.19) 95.1 (4.16) York-S 0.0 (0.00)  7.2 (5.26) 92.8 (5.26)

In Rochelle-US, there was no visible generational trend for change inthe rate larval development as a potential result of inbreedingdepression or genetic drift that may have resulted from bottleneckingthis colony each generation. Rochelle-S on DAS-59122-7 produced morelarvae in later instars compared to the corresponding Rochelle-US colonyon DAS-59122-7 in every generation except F3 (P=0.57) (Table 2). Thischange in the rate of larval development is indicative of selecting forincreased WCR tolerance to DAS-59122-7. Tolerance in Rochelle-S wasapparent after the first round of selection (F1). After 17 days, thepercentage of first and second instars in Rochelle-US was 76.0 and 17.3%compared to 46.7 and 48.0% in Rochelle-S. After F1, tolerance inRochelle-S to DAS-59122-7 appeared variable around an intermediate level(Table 2). A high level or complete Rochelle-S resistance to DAS-59122-7might have resulted in SSA results similar to Rochelle-US or Rochelle-Scolony development on isoline (Table 4). Instead, Rochelle-S resulted inan across-generation average of 49.8% of larvae in the third instar whenexposed to DAS-59122-7 instead of the 86.2 or 81.6% measured whenRochelle-US and Rochelle-S were exposed to isoline maize, respectively(Table 2). After F1, there was no consistent trend for increaseddevelopmental rate of Rochelle-S on DAS-59122-7 with each generation ofselection.

In York-US, there was no visible generational trend for change in therate of larval development as a potential result of inbreedingdepression or genetic drift that may have resulted from bottleneckingthis colony each generation. In every generation, York-S on DAS-59122-7produced more larvae in later instars compared to the correspondingYork-US colony on DAS-59122-7 (Table 3). Tolerance in York-S wasapparent after the first round of selection (F1). After 17 days, thepercentage of first and second instars in York-US was 82.7 and 10.7%compared to 70.7 and 28.0% in York-S. After F1, tolerance of York-S toDAS-59122-7 appeared variable around an intermediate level. A high levelor complete York-S resistance to DAS-59122-7 might have resulted in SSAresults similar to York-US or York-S colony development on isoline(Table 4). Tolerance may have been greatest in the F3 generation where85.3% of larvae were able to develop to third instar compared to theacross-generation average of 53.3%. Tolerance in York-S is intermediategiven the across generation mean number of larvae in third instar is53.3% using DAS-59122-7 compared to 95.1 and 92.8% for York-US andYork-S using isoline (Table 4).

Estimates of heritability (h²) are presented for the Rochelle-S andYork-S colonies in FIG. 1. Realized heritability was estimated at 0.31and 0.27 for the F1 generation of Rochelle-S and York-S, respectively.After 10 and 11 generations, realized heritability was estimated at 0.10and 0.11 for Rochelle-S and York-S, respectively (FIG. 1). There was anoverall trend for a decline in realized heritability in both Rochelle-Sand York-S with successive generations of selection and it appeared thelargest reduction in variation occurred during the first fewgenerations.

The impact of selection was detectible in the F1 generation ofRochelle-S. Survival rate of the F1 generation on DAS-59122-7 was 13%compared to the Rochelle-US across generation mean of 76.2% on isoline(Table 1). The percentage of females was lower in the Rochelle-S F1generation compared to Rochelle-US and the Rochelle-S females weregenerally less fecund (Table 1). There were no apparent differences infertility between F1 eggs of Rochelle-US and Rochelle-S colonies. Meansurvival rate of the Rochelle-S colony increased gradually over thefirst five generations (Table 1). Survival rate in the F5 progeny was15.9% or 5.1% higher than the across generation Rochelle-S mean (10.8%).After F1, estimates of percentage female, fecundity and fertility werevariable around the across generation mean for each trait. There was noobvious across-generational trend in other life history traits measuredfor the Rochelle-S colony.

Similarly, the impact of selection was detectible in the F1 generationof York-S. Survival rate of the F1 generation on DAS-59122-7 was 0.4%compared to the York-US across generation mean of 82.7% on isoline maize(Table 1). The percentage of females was lower in the York-S F1generation compared to York-US and York-S females were generally lessfecund (Table 1). There was no apparent difference in fertility betweenF1 eggs of York-US and York-S colonies. Mean survival rate of the York-Scolony was variable around an across generation mean of 10.3% (Table 1).Survival rate on DAS-59122-7 increased from 0.4 to 8.5% between the F1and F2 generations; however 3 out of 9 subsequent generations resultedin survival rates less than 8.5% and the maximum survival rate was 23.4%in the F9 progeny. After F1, estimates of survival rate, percentagefemale, fecundity and fertility were variable around the York-Sacross-generation means for each trait. There was no obvious acrossgenerational trend in any adult life history traits measured for theYork-S colony.

The root trainer experiment testing the injury potential of the F1 andF2 generations of Rochelle-S on DAS-59122-7 was repeated over 6 dates.When data were pooled from this experiment, there was a significant dateby treatment interaction (F=3.4; df=15,54; P=0.0005) resulting from themean node-injury score for the first date (0.11). This node-injury scorerepresented 50% of the injury level observed for the other 5 dates(0.20-0.29). Data from the first date were then removed from the pooledanalysis, resulting in a non-significant date effect (F=1.7; df=4.15;P=0.20) and date*treatment interaction (F=0.75; df=12.45; P=0.69).Across the 5 remaining dates, treatment had a significant effect on meannode-injury scores (F=233.7; df=3.12; P<0.0001). The mean node-injuryscore for Rochelle-US on isoline (0.76) was significantly greater thanthe other 3 treatments and indicated a moderate level of feedingpressure in this experiment (Table 5). There was no significantdifference in mean node-injury scores between the F1 and F2 generationsof Rochelle-S on DAS-59122-7 (Table 5). There was, however, a subtlenumerical increase in mean node-injury measured for both the F1 and F2generations of Rochelle-S on DAS-59122-7 compared to Rochelle-US onDAS-59122-7, but neither increase was statistically significant (Table5). This subtle increase in injury potential to DAS-59122-7 was alsoevident in the consistency ratings, where the percentage of roots in the0.26-0.50 node-injury category was 0% for Rochelle-US on DAS-59122-7compared to 1.7 and 4.3% for the F1 and F2 generations of Rochelle-S,respectively. Table 5 illustrates the number of viable eggs infested perplant, mean node-injury ratings, node-injury scores per 100 viable eggs,and percentage of roots in 5 node-injury categories for greenhouseefficacy experiments characterizing the injury-potential of theRochelle-S and Rochelle-US colonies on event DAS-59122-7 andnontransgenic isoline maize across generations of selection onDAS-59122-7.

The injury potential of Rochelle-S was evaluated in 2 additional largepot experiments; at generations F5 and F10 (Table 5). In bothexperiments, treatment had a significant effect on mean node-injuryscores (F5: F=156.2; df=3.12; P<0.0001 and F10: F=64.2; df=3.12;P<0.0001). The mean node-injury scores for Rochelle-US on isoline (2.57for F4 and 2.70 for F10) indicated a very high level of feeding pressurewas achieved relative to the initial root trainer experiment (Table 5).Mean node-injury scores for the F5 and F10 generations of Rochelle-S onDAS-59122-7 were also significantly greater compared to thecorresponding generations of Rochelle-US on DAS-59122-7. (Table 5). Themean node-injury score for Rochelle-S on isoline was significantly lessthan Rochelle-US on isoline at F5. However, at F10, Rochelle-S andRochelle-US caused a similar amount of injury on isoline maize (Table5).

TABLE 5 Genera- tion Viable Node- of eggs Mean ± SE injury Experi-selec- Maize infested/ node-injury rating/100 Percentage of roots ineach node-injury category ment¹ tion Insect colony hybrid n plant²rating³ eggs 0.00-0.25 0.26-0.50 0.51-0.99 1.00-1.99 2.00-3.00 TrainerF1 Rochelle-S 59122 115 39 0.07 ± 0.020 a 0.18 98.3 1.7 0 0 0 F2Rochelle-S 59122 117 41 0.08 ± 0.019 a 0.19 95.7 4.3 0 0 0 F2Rochelle-US 59122 112 41 0.03 ± 0.020 a 0.07 100 0 0 0 0 F2 Rochelle-USIsoline 119 41 0.76 ± 0.019 b 1.85 16.8 14.3 24.4 43.7 0.8 Pot 1 F5Rochelle-S 59122 49 484 0.85 ± 0.091 a 0.18 26.5 18.4 18.4 24.5 12.2 F4Rochelle-US 59122 50 470 0.10 ± 0.090 b 0.02 94 4 2 0 0 F5 Rochelle-SIsoline 50 484 2.29 ± 0.090 c 0.47 2 2 4 16 76 F4 Rochelle-US Isoline 50470 2.57 ± 0.090 d 0.55 2 2 2 8 86 Pot 2 F10 Rochelle-S 59122 30 7261.83 ± 0.102 a 0.25 6.7 3.3 13.3 26.7 50 F10 Rochelle-US 59122 30 8040.19 ± 0.102 b 0.02 79.9 6.7 6.7 6.7 0 F10 Rochelle-S Isoline 30 7262.89 ± 0.102 c 0.40 0 0 0 3.3 96.7 F10 Rochelle-US Isoline 30 804 2.70 ±0.102 c 0.34 3.3 0 0 0 96.7 Within each experiment, means followed bythe same lower-case letter are not significantly different at P ≦ 0.05(t-test). ¹Three greenhouse efficacy experiments were conducted usingeither a root trainer or large pot test system. ²Plants in the trainerexperiment infested with 100 eggs 13 days after planting. Plants in thePot 1 experiment infested with 400 and 1,000 eggs 17 and 24 days afterplanting (1,400 eggs total). Plants in the Pot 2 experiment infestedwith 600 eggs 14 and 20 days after planting (1,200 eggs total). Hatchtests were used to estimate number of viable eggs infested. ³Roots werevisually scored for injury using the 0-3 node-injury scale (Oleson etal. 2005). Roots scored 32 days after planting in the trainerexperiment, 59 days after planting in the Pot 1 experiment, and 60 daysafter planting in the Pot 2 experiment.

A similar change in injury potential was measured for the F1 and F2generations of York-S on DAS-59122-7 in the large pot test system. Inthis experiment, treatment also had a significant effect on meannode-injury scores (F=18.2; df=3.12; P<0.0001). Table 6 describes thenumber of viable eggs infested per plant, mean node-injury ratings,node-injury scores per 100 viable eggs, and percentage of roots in 5node-injury categories for greenhouse efficacy experimentscharacterizing the injury-potential of the York-S and York-US colonieson DAS-59122-7 and nontransgenic isoline maize across generations ofselection on DAS-59122-7. The mean node-injury score for York-US onisoline (0.62) was significantly greater than the other 3 treatments andindicated a moderate level of feeding pressure in this experiment (Table6). There was no significant difference in mean node-injury scoresbetween York-S F1 and F2 (Table 6). However, both the F1 and F2generations caused significantly more root injury to DAS-59122-7compared to York-US on DAS-59122-7 (Table 6). However, at F10,Rochelle-S and Rochelle-US caused a similar amount of injury on isolinemaize (Table 5). This significant increase in injury potential after 1and 2 generations of selection was also evident in the consistencyratings, where the percentage of roots in each node-injury categoryshifted to higher categories with each additional generation ofselection (Table 6).

The injury response for York-S at generations F6 and F11 was nearlyidentical to that observed for Rochelle-S at generations F5 and F10. Inthe experiments evaluating York-S F6 and F11, treatment had asignificant effect on mean node-injury scores (F6: F=147.9; df=3.12;P<0.0001 and F11: F=179.2; df=3.12; P<0.0001). Mean node-injury scoresfor the F6 and F11 generations of York-S on DAS-59122-7 were alsosignificantly higher than the corresponding F4 and F10 York-US scores onDAS-59122-7 (Table 6). The mean node-injury score for York-S F6 onisoline was significantly greater than York-US F4 on isoline at F5.However, at F11, Rochelle-S and Rochelle-US caused a similar amount ofinjury on isoline maize (Table 5).

TABLE 6 Genera- Viable tion eggs Mean ± SE Node-injury Experi- of InsectMaize infested/ node- rating/100 Percentage of roots in each node-injurycategory ment¹ selection colony hybrid n plant² injury rating³ eggs0.00-0.25 0.26-0.50 0.51-0.99 1.00-1.99 2.00-3.00 Pot 3 F1 York-S 5912255 136 0.11 ± 0.031 a 0.08 89.1 7.3 3.6 0 0 F2 York-S 59122 45 134 0.19± 0.035 a 0.14 75.6 11.1 11.1 2.2 0 F1 York-US 59122 55 144 0.01 ± 0.031b 0.01 100 0 0 0 0 F1 York-US Isoline 55 144 0.62 ± 0.031 c 0.43 29.114.5 29.1 27.3 0 Pot 4 F6 York-S 59122 50 622 0.52 ± 0.077 a 0.08 36 2810 26 0 F4 York-US 59122 50 429 0.03 ± 0.077 b 0.01 50 0 0 0 0 F6 York-SIsoline 50 622 2.37 ± 0.077 c 0.38 2 0 0 20 78 F4 York-US Isoline 50 4291.02 ± 0.077 d 0.24 20 16 12 34 18 Pot 5 F11 York-S 59122 30 726 2.07 ±0.109 a 0.28 3.3 6.7 10 23.3 56.7 F10 York-US 59122 30 756 0.13 ± 0.109b 0.02 86.6 6.7 6.7 0 0 F11 York-S Isoline 30 726 2.85 ± 0.109 c 0.39 00 0 3.3 96.7 F10 York-US Isoline 30 756 2.89 ± 0.109 c 0.38 0 0 0 0 100Within each experiment, means followed by the same lower-case letter arenot significantly different at P ≦ 0.05 (t-test). ¹Three greenhouseefficacy experiments were conducted using a large pot test system.²Plants in the Pot 3 experiment infested with 100 eggs 13, 19, and 28days after planting (300 eggs total). Plants in the Pot 4 experimentinfested with 400 and 1,000 eggs 17 and 24 days after planting (1,400eggs total). Plants in the Pot 5 experiment were infested with 600 eggs14 and 20 days after planting (1,200 eggs total). Hatch tests were usedto estimate number of viable eggs infested. ³Roots were visually scoredfor injury using the 0-3 node-injury scale (Oleson et al. 2005). Rootsscored 74 days after planting in the Pot 3 experiment, 59 days afterplanting in the Pot 4 experiment, and 60 days after planting in the Pot5 experiment.

The mean node-injury score per 100 viable eggs was calculated for eachtreatment across experiments to better facilitate comparison of injurypotential to DAS-59122-7 across generations of selection. This metricdoes not account for intraspecific competition effects; however, it doesallow for a normalized comparison of injury potential across experimentswith different rates WCR egg infestation. For both unselected colonies,the mean node-injury score per 100 viable eggs on DAS-59122-7 remainedconsistently low across generations, ranging from 0.01 to 0.07 (lessthan 1 root pruned) (Tables 5 and 6). For Rochelle-S, the meannode-injury score per 100 viable eggs on DAS-59122-7 was 0.18, 0.19,0.18, and 0.25 for generations F1, F2, F5, and F10, respectively (Table5). A similar response was observed for York-S on DAS-59122-7 acrossgenerations, where the mean node-injury score per 100 viable eggs onDAS-59122-7 was 0.08, 0.14, 0.08, and 0.28 for generations F1, F2, F6and F11 (Table 6). Results from both selected colonies indicate thatsurvivors from the first generation of selection have a slight increasein injury potential and that repeated generations of selection onDAS-59122-7 without random mating resulted in a gradual increase in theinjury potential to DAS-59122-7.

It is noteworthy that these colonies exhibit tolerance, rather thancomplete resistance, to event DAS-59122-7. In this respect, thedevelopment of the colonies has already provided valuable information,namely that a single gene that is responsible for complete resistance toDAS-59122-7 is rare in wild populations, that a heritable characteristicconferring tolerance to DAS-59122-7 is present in wild populations, thatthis heritable characteristic is not a realistic threat to thedurability of DAS-59122-7 efficacy. Instead, based on these data, a morecomplex genetic basis for intermediate tolerance to DAS-59122-7 isapparently present in WCR populations, with expression of one ormultiple genes resulting in intermediate tolerance to the event.Tolerance was heritable, and results from consecutive generations of theselection show the tolerance is variable around an intermediate level,the incidence of these partial resistance gene or genes appears variablebut stable over several generations of selection without random mating,thereby resulting in variable levels of tolerance and larval injurypotential to DAS-59122-7 maize over several generations.

The knowledge that a major resistance gene in WCR to DAS-59122-7 is rarein wild populations presents several opportunities for investigation anduse of colonies tolerant to the event.

One potential use is in the realm of negative cross-resistance (NCR).The current generation of pesticides includes toxins isolated frombacterial broths, such as Spinosad, and transgenic plants containinggenes that code for an insecticidal protein. It is highly likely that insome cases target-site insensitivity to these new classes ofinsecticides occur in the pest insects. Target-site insensitivity is amajor mechanism of resistance to second generation pesticides. Afterdeploying these novel toxins, it is likely that a single (or multiple)point mutation in the gene coding for the target site in the insect'sgut or other target system results in the insects developing fieldresistance. Additionally, metabolic resistance may occur where theinsects have a greater ability to alter the toxin such that it hasreduced toxic activity.

Even if metabolic resistance occurs to such resistance factors, themetabolic resistance does not rule out the possibility of developing NCRcompounds for control of metabolic insecticide resistance.

In spite of the lack of large-scale screening for NCR toxins, therestill has been discovery of such compounds. For example, a NCR factor toaphids has been identified that was resistant to insecticides throughincreased production of a carboxylesterase, E4.

Although NCR factors do occur within classes of toxins, there is nodistinct reason to believe that NCR factors will only be found in thesame class of compounds as the first toxin. Although compounds withinthe classes of toxins appear to be a logical starting place, exemplaryscreens for NCR toxins involve random screens for compounds. The randomscreens are, in one embodiment, coupled with a “clue-based” screen.

An advantage of random screening for NCR factors is that anunderstanding of the molecular basis of resistance is not necessary forthe development of the second compound. Knowledge on the molecular basisof resistance typically lags years behind the first appearance oftolerant insects in the field. However, knowing the basis of toleranceis helpful for ‘clue-based’ screening. But if discovery of the molecularbasis of pesticide tolerance is too costly or time consuming, one may beable to use the tolerant line (or lines) in a random screen for NCRfactors.

Tests using tolerant and susceptible lines of insects are easilyintegrated into current large-scale automated screening methodologies.The screens identify compounds that are toxic to the tolerant line (orlines) in the bioassay and not toxic to the insect lines that aresusceptible to the already commercialized toxin.

Ordinarily when seeking negative cross-resistance in pest controlcompounds, the mechanism of resistance is based on a single gene,meaning a homozygous resistant strain (R/R) is evaluated with a numberof potentially toxic molecules, e.g., natural molecules, syntheticmolecules, chemicals, compounds, biotechnical species, and biotechnicalmoieties, to determine a second toxin that is more toxic to theresistant strain (R/R) than to the susceptible strain (S/S). The toxicmolecules include variants, mutants, metabolites, and derivatives. Asusceptible control strain (S/S) is also evaluated with the samecompounds. Exemplary chemicals include a) Bacillus thuringiensisproteins and their variants, b) clorinated hydrocarbons, c)organophosphates, d) pyrethroids, e) carbamates, f) variants of toxinsfrom the bacteria Photorhabdus luminescens, g) insect growth regulatorsand their derivatives, h) alpha-amylase inhibitors, i) lectins, j)Spinosad derivatives, k) spinosyns and their derivatives, l) derivativesof insecticidal compounds from the bacteria Saccharopolyspora spinosa,m) Bacillus thuringiensis strains and their variants, n) proteaseinhibitors and their derivatives, o) Cysteine protease inhibitors andtheir derivatives, p) Bowman-Birk Inhibitors and their derivatives, q)Kunitz inhibitors and their derivatives, r) Saccharopolyspora spinosastrains and derivatives of their insecticidal and non-insecticidaltoxins, and s) imidacloprid or derivatives of imidacloprid.

Molecules may be supplied from randomly or selectively generatedchemicals, and random or selective (chemical rationale approach)screening of chemicals. The molecules to be evaluated further includemolecules supplied from bio-prospecting from plant, animal, bacteria,and fungal organisms or extracts of these organisms and from prokaryoticor eukaryotic organisms. The molecules to be evaluated also includemolecules supplied from the generation of antibodies showing preferencefor binding to proteins or protein complexes or membranes in theorganism involved in negative cross-tolerance (binding preference forversions of the protein that are resistant to the first toxin) andgeneration of random peptide libraries and bio-panning using phagedisplay. A random peptide library is made and is screened for affinityto the product of the target of interest, e.g., the gene product of thetarget site. The resistant allele, more specifically the proteinproduct, is then used to identify a protein that has high affinity tothe gene product to generate a NCR toxin for specifically targeting theresistant insect. The molecules to be evaluated also include moleculesobtained from combinatorial shape libraries and molecules supplied usingcombinatorial chemistry. Those compounds that are more toxic to theresistant strain than to the susceptible strain are considered to bepositive compounds for the initial evaluation.

A heterozygous strain (R/S) of the target population is evaluated withthe positive compounds to test their effectiveness against theheterozygous insects. Thus, the resistant (R/R) and susceptible (S/S)insects are crossed and the progeny bio-assayed against the new toxin.It should be determined whether tolerance is sex-linked, since if thetolerance is sex-linked, individuals of the proper sex that carry twoalleles of the gene should be used. The heterozygotes are screened byusing separate applications of the first toxin and the positive compoundbeing tested, i.e., the second toxin, to determine if separateapplications of the first toxin and the second toxin are at least astoxic to the heterozygous strain (R/S) as to the susceptible strain(S/S) of the target population.

If the heterozygotes are killed by separate applications of the firsttoxin and the positive compound being tested, the positive compound isgiven a high priority for development and commercial exploitation. Ahigh negative cross-tolerance priority is assigned to the second toxinif separate applications of the first toxin and the second toxin are atleast as toxic to the heterozygous strain (R/S) as to the susceptiblestrain (S/S). Thus, based on the toxicity of the compound toheterozygous individuals, the practical applicability of each toxin isprioritized and the compounds capable of killing the heterozygotesreceive a high priority while those compounds that only impacthomozygous individuals are subjected to further testing and evaluationto determine their prioritization. The priority compounds, in oneembodiment, are prioritized for advancement to additional evaluationswhich are utilized to make commercial development prioritizationdecisions. In an alternative embodiment, the high priority compoundsreceive a commercialization prioritization.

The heterozygous strain is evaluated with the first toxin and the secondtoxin applied at the same time to determine if the application of thefirst toxin and the second toxin at the same time is at least as toxicto the heterozygous strain (R/S) as to the susceptible strain (S/S). Ifthe application of the first toxin and the second toxin at the same timeis not at least as toxic to the heterozygous strain as to thesusceptible strain, the compound is given a low priority for furtherdevelopment.

In the case of insects tolerant to DAS-59122-7, including but notlimited to the York and Rochelle colonies, however, the mechanism ofresistance appears to be more complex. As noted above, based on the dataproduced during selection of the tolerant colonies, it appears that thistolerance to event DAS-59122-7 is not a simple gene model where a singlerecessive gene with two alleles confers resistance to the event. Rather,this mechanism of tolerance to DAS-59122-7 appears to be a more complexgenetic system comprising multiple genes. This is because despite manygenerations of selection for tolerant organisms, the tolerant strainsstill do not exhibit complete or near-complete resistance to the event.This is in contrast to the mechanism of resistance for most other knownpesticides and herbicides, where frequently a mutation at a single geneis sufficient to confer resistance.

The nature of the tolerance to DAS-59122-7, therefore, presentsadditional opportunities in the context of insect resistance management.For example, while ordinarily negative cross-resistance is a binarysituation (either an organism has resistance, and therefore negativecross-resistance, or it does not), with the more complex tolerancegenetics of resistance to DAS-59122-7, there are potentially a greaternumber of possible negative cross-resistance situations. This is becausea rootworm that has greater susceptibility to DAS-59122-7 may, dependingon the basis of the additional susceptibility, have increasedsusceptibility to one or more other toxins. Given that there aremultiple genes for which negative cross-resistance (or tolerance) may beassociated, it is possible for many negative cross-resistant compoundsto either exist currently or be engineered based on the mechanism oftolerance to DAS-59122-7.

Once a pair of NCR factors is determined, many different types ofapplications of the toxins to the insects can be used. For example, bothtoxins can be applied at the same time every time, one of the toxins canbe applied on an intermittent basis, both toxins can be applied on anintermittent basis, and the toxins can be applied in an alternating typeapplication. In one embodiment, the toxins are delivered to the targetpopulation utilizing at least one of sprays, pellets, powders, baited ornon-baited traps, and transgenic organisms. For example, in the case ofwestern corn rootworm, a first compound is introduced to the field byspraying the compound on the field, incorporating the compound viatransgenic crops, or any other method known in the art. In the contextof DAS-59122-7, typically transgenic corn incorporating that event willbe combined with another form of pesticide, whether it be chemical,transgenic, or some other form. If resistant forms exist in theparticular field, a second compound is then applied to the field. Inaddition, transgenic antibodies or antibody conjugates with toxinsattached could be used in the selection assays. Thus, in one embodiment,the above-described method is used to manage a tract of land against aresistant strain of a target population. In an alternative embodiment,the above described method is used as part of a pest management systemto manage a pest population.

Further detail regarding methods for screening for negativecross-tolerance may be found in PCT Publication WO 01/92561 A2, hereinincorporated by reference in its entirety. In addition to usefulness andnegative cross-tolerance development strategies, the colonies of thepresent invention are also useful in understanding the mechanism ofwestern corn rootworm tolerance to various insect control strategies.The resistant colonies may be compared to wild type colonies lackingsuch tolerance or containing such tolerance at lower frequencies todetermine genetic and/or phenotypic differences between such insects,thereby assisting in identification of the tolerance mechanism in theselected colonies.

This has the potential to be particularly helpful given the complexgenetic nature of tolerance to DAS-59122-7. Genetic comparisons betweenthe tolerant colonies disclosed herein and susceptible or wild type WCRcolonies will yield valuable information about both the nature of theDAS-59122-7 event itself as well as the mechanism by which insects buildtolerance to the event. This type of analysis may be done by any numberof methods known in the art.

Further, these colonies may be used to determine whether positivecross-tolerance exists with any currently-existing corn rootworm controltactics. For example, the selected colonies may be exposed to maizeplants exhibiting transgenic events such as MON863, Cry3Aa, other Cryproteins, or chemical insecticides, to determine whether positivecross-tolerance exists. In addition to determining whether suchcross-tolerance exists, it will also help determine the potential for,and estimated rate of, field development of tolerance to eventDAS-59122-7.

Another opportunity in the context of resistance management is using thetolerance traits identified via the York and Rochelle selected coloniesas part of a resistance management strategy protecting against recessivetraits conferring resistance or tolerance levels greater than thecharacteristics of the York or Rochelle selected colonies. The mostfrequently-used current IRM strategy is exposing insects to a high doseof a pest control substance and the planting of a refuge (a portion ofthe total acreage using seed lacking a gene conferring pest resistance),as it is commonly-believed that this will delay the development ofinsect resistance to resistant crops by maintaining insectsusceptibility. The high dose/refuge strategy assumes that resistance isrecessive and is conferred by a single locus with two alleles resultingin three genotypes: susceptible homozygotes (SS), heterozygotes (RS),and resistant homozygotes (RR). It also assumes that there will be a lowinitial resistance allele frequency and that there will be extensiverandom mating between resistant and susceptible adults. Under idealcircumstances, only rare RR individuals will survive a high doseproduced by the resistant crop or otherwise exposed to the pests. BothSS and RS individuals will be susceptible to the given toxin. Astructured refuge is a non-pesticidal portion of a grower's field or setof fields that provides for the production of susceptible (SS) insectsthat may randomly mate with rare resistant (RR) insects surviving thepesticidal crop to produce susceptible RS heterozygotes that will bekilled by the pesticidal crop. This will remove resistant (R) allelesfrom the insect populations and delay the evolution of resistance.

The high dose/refuge strategy is the currently-preferred strategy forIRM. Non-high dose strategies are currently used in an IRM strategy byincreasing refuge size. The refuge is increased because lack of a highdose could allow partially resistant (i.e., heterozygous insects withone resistance allele) to survive, thus increasing the frequency ofresistance genes in an insect population. For this reason, numerous IRMresearchers and expert groups have concurred that non-high doseexpression of insecticidal traits presents a substantial resistance riskrelative to high dose expression. However, such non-high dose strategiesare typically unacceptable for the farmer, as the greater refuge sizeleaves a larger proportion of the crop at risk to greater pest damageand further loss of yield.

Currently, the size, placement, and management of the refuge is oftenconsidered critical to the success of the high dose/structured refugestrategy to mitigate insect resistance to the Bt proteins produced incorn, cotton, and potatoes. Structured refuges are generally required toinclude all suitable non-pesticidal host plants for a targeted pest thatare planted and managed by people. These refuges could be planted tooffer refuges at the same time when the resistant crops are available tothe pests or at times when the resistant crops are not available. Theproblems with these types of refuges include ensuring compliance withthe requirements by individual farmers. Because of increased pestpressure and the decrease in yield in refuge planting areas, somefarmers choose to eschew the refuge requirements, and others do notfollow the size and/or placement requirements. These non-complianceissues result in either no refuge or less effective refuge, and acorresponding increase in the development of resistant pests.

A prominent hypothesis is that minor insecticide resistance or tolerancegenes can accelerate pest adaptation via major resistance genes. Thishas been referred to as coadaptation; where selection and integration ofresistance genes with other loci ameliorate the deleterious effects ofresistance. The general theory goes that increased pest survival from aminor gene creates a means by which rare major resistance genes escapeselection and thereby increase in frequency. This hypothesis is basedmostly on the early work using pest exposure to synthetic organicinsecticides. A classic example of this interaction is the PEN—a gene,which slows cuticular penetration of synthetic insecticides inhouseflies. In this instance, houseflies developed resistance based on a(minor) cuticle gene and not a major putative resistance gene thatmetabolizes the toxin or confers a receptor-mediated resistance etc.More recently, laboratory selection experiments have been used to studythe interactions between polygenic “minor” and monogenic “major” genesfor pest resistance. In laboratory selection experiments, the rate ofinsect exposure to insecticides are often reduced well below fieldexposure rates. This scenario facilitates experiments with smallpopulations that may be otherwise killed by exposure to field rates ofinsecticide. In laboratory experiments, tolerant or resistant phenotypesfrequently result from polygenic traits, compared to monogenic traitsidentified in the same species where selection in the field occurs atsignificantly higher rates. The precision with which laboratoryselection results can be extrapolated to field conditions is discussedby Groeters and Tabashnik (2000). They use an analysis ofliterature-based data along with a simulation model to test hypotheseson whether the method of selection biases selection experiment resultstoward major or minor genes. They found little association between thetype of selection method and whether the method yielded major or minorresistance genes. Results of their simulation modeling showed theeffectiveness of refuge was related more to the intensity of selectionand less to whether resistance was considered major or minor. Theyconclude that understanding selection intensity is more important thanunderstanding the number and relative contribution of resistance loci. Amuch less popular hypothesis is that minor or polygenic resistancetraits can interfere with pest adaptation via major monogenicresistance. Lande (1983) describes how pest adaptation via a majorresistance gene (monogenic mutation) can be prevented or delayed by aminor (polygenic mutation) resistance gene despite strong selectionpressure; rarity of major resistance is an essential condition for thisinteraction.

This interaction may be present in the DAS-59122-7 maize and cornrootworm system and could have a large and beneficial effect on thedurability of DAS-59122-7 maize. The characteristic identified in theYork and Rochelle selected colonies creates resistance managementopportunity in that rootworms possessing these genes conferringtolerance, without conferring complete or major resistance, can act asrefuge insects. As a result, crops incorporating DAS-59122-7 should beable to be planted with little or no refuge plants for rootworms, as thenature of the development of resistance to the event is contrary to thetraditionally-believed resistance paradigm.

The potential effectiveness of this resistance management strategy isillustrated by FIG. 2. In FIG. 2, as described previously, the expecteddevelopment of resistance to DAS-59122-7 is indicated by the lines,given either a 0.0005 or 0.001 incidence of a resistance allele presentin the wild. As can be seen from the data from generations of selectionof the York and Rochelle tolerant colonies, resistance has not developedaccording to the predicted timeframe. Instead, a low level of toleranceto DAS-59122-7 has developed over the course of generations, and hasvaried somewhat as opposed to steadily increased. This is consistentwith a polygenic tolerance mechanism, where certain genes contributingto tolerance are lost from one generation to the next due to variousgenetic events, such as crossing over or mutations.

Several factors associated with the selection materials and methodsincrease the inferential power of findings from this study. First, theselection likely included a large degree of existing additive geneticvariation for DAS-59122-7 tolerance. Each founding population of WCRoriginated with 1,000 wild males. The work of Kim et al. (2007) suggeststhat there was very little loss of genetic diversity by introgressing anon-diapause trait into the Rochelle, Ill. and York, Nebr. foundingpopulations. In a genetic analysis of several diapausing and onenon-diapausing laboratory colonies, they found relatively low geneticdiversity among nine diapausing colonies that had been reared forapproximately 0-22 generations and only a moderate loss of geneticdiversity in the non-diapausing colony that had been reared forapproximately 190 generations. Although Rochelle, Ill. and York, Nebr.are separated by approximately 800 km, results from this study suggestthere was a similar degree of additive genetic variation for toleranceto DAS-59122-7 in both Rochelle, Ill. and York, Nebr. foundingpopulations. These results corroborate the earlier work usingmicrosatellite markers to study genetic variation in rootwormpopulations from Kansas to the east coast of the US, which concludedthere was high genetic similarity in WCR over much of the US and that noobvious genetic structuring had resulted since the eastward expansion ofthis pest. As a result, the information gleaned from these experimentsand data is more broadly applicable to US WCR populations and notspecific to the regions surrounding Rochelle, Ill. or York, Nebr., andstrategies developed from this information have broader applicability.

Further, the method of selection was ecologically relevant. Larvalexposure to insecticidal proteins in the roots of maize is complex overspace and time, and root-tissue function is the primary explanation forvariation in total protein. For example, the growing point ismetabolically active, undergoing rapid cell division, protected bymucilage and relatively rich in total soluble protein compared to moredistal tissue that has mostly structural and vascular functions. Therelative quantity and distribution of insecticidal protein made byDAS-59122-7 follows the pattern of total soluble protein in maize roots.Consequently, using DAS-59122-7 as a means of selection increased thelikelihood of integrating realistic plant and larval interactionsresulting when exposure is complex. Moreover, similarity between the F1survival rates of 0.4 to 1.3%, using seedlings and laboratoryconditions, and the field estimates of 0.6 to 4.0% reported by Storer etal. (2006) shows there was a high degree of ecological relevance in theselection method. The fact that these data represent a closerapproximation to actual field conditions as compared to direct-exposurestudies also support the more general applicability of data obtainedfrom these tolerant colonies, as well as the resistance managementstrategies and other information that can be derived from the tolerantinsects.

It can be concluded from this resistance risk assessment that putativeor major resistance to DAS-59122-7 is rare in US populations of westerncorn rootworm. Increased confidence comes from the large size of initialcollections relative to the known genetic diversity in rootwormpopulations and the ecological relevance of the selection method.Survival of rootworms on DAS-59122-7 in the laboratory and the field isvery low and variable across locations and years. It can be concludedthat much of the rootworm survival on DAS-59122-7 is heritable andinheritance of the apparently polygenic tolerance trait is complex. Thistolerance trait is considered minor as it relates to the efficacy ofDAS-59122-7.

As can be seen from these data, even with no refuge insects to dilutethe presence of genes conferring resistance or tolerance to DAS-59122-7,the York and Rochelle selected colonies had not developed putativeresistance to the event, even after at least 10 generations ofselection. As also can be seen from these data, WCR that surviveDAS-59122-7 have a heritable trait or traits that appear independent ofa theoretical major single gene for resistance to DAS-59122-7 withrecessive inheritance. Consequently, insects with this characteristicidentified in Rochelle and York selected colonies can serve as refugefor major resistance genes for other pest management strategies. Giventhese data, plants incorporating DAS-59122-7 should not need astructured refuge in order to prevent resistance to the event fromdeveloping, or at a minimum, a drastically reduced refuge (as comparedto the currently-accepted 20%) may be used. For example, plantsincorporating DAS-59122-7 may comprise 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or even 100% of theplants intended to be in a given plot. Of course, low levels of off-typeseeds can occur normally during commercial hybrid seed production, so aplot will rarely, if ever, actually have 100% of its plantsincorporating event DAS-59122-7. As a result, a plot may be consideredto have 100% of its plants incorporating this event even if some lowlevel of seeds not containing event DAS-59122-7 are present.

The ability to reduce or eliminate structured refuge for this event willresult in an increase in crops protected from rootworm infestation byDAS-59122-7, produce a substantial pool of susceptible rootworms todelay major gene resistance, and accordingly increased yields forgrowers. It also reduces or eliminates the issues regarding compliancethat are inherent whenever a structured refuge is required. As a result,a grower could have the entirety of a given crop protected withDAS-59122-7 to deter rootworms, as well as an additional 80% of the crop(or whatever percentage is required from a regulatory perspective)protected with an alternative pest management strategy, such as chemicalpesticide, another transgenic event, or any other pest managementmethod, in order to improve control of rootworms further while stillmaintaining the commonly-used structured refuge for the second pestmanagement strategy.

Simulation data supports the empirical data described above, namely thata tolerance trait can produce sufficient refuge insects to delay thedevelopment of resistant insects. A simple population genetics model ofDiabrotica in a landscape of continuous corn was used to explain how thetolerance trait(s) can act as an adaptive barrier or slow thedevelopment of resistance based on major resistance genes. Twoautosomal, di-allelic, genes are modeled: a minor (tolerance) genedesignated with X for wild type and Y for tolerance; and a major genedesignated with A for wild type and B for resistance, with a maximumsurvival of 1.0 on toxic corn. For the purpose of this simulation model,rootworm tolerance to event DAS-59122-7 was assumed to be a single geneand parameter estimates for this gene were derived from the biologicalresponse of the surviving phenotype reported in Lefko et al. (2008),which is incorporated by reference herein in its entirety. To study theinteraction between the tolerance trait and putative resistance; WCRadaptation to DAS-59122-7 was first modeled without the tolerance traitor Y allele. We excluded the Y allele, used off-type seeds at aproportion 0.0075 and no structured refuge. We define off-types here asseeds not containing DAS-59122-7. Results of this simulation arepresented in FIG. 3.

Putative resistance developed rapidly with the proportion ofheterozygous (AB) individuals peaking at 0.3 after 5 generations andhomozygous (BB) individuals exceeding 0.5 after 6 generations (FIG. 3).The line predicting the increase in the proportion of homozygous (BB)resistant individuals was very steep with 0.69 of the populationhomozygous for B in 6 generations and reaching 0.99 in 8 generations.

The effect of a 20% structured adjacent block refuge excluding the Yallele (FIG. 4) and using the same parameters as used in FIG. 3 was thensimulated. This simulation exemplifies the currently-accepted resistancemanagement strategy for events with putative or major resistancegene(s). The relative impact of the 20% block refuge was large; itdelayed the onset of ≧0.5 B allele frequency by roughly 2.5 times withthe heterozygous (AB) proportion of the population peaking at 0.21 after16 generations and the homozygote proportion reaching 0.85 after 17generations. The line predicting the proportion of homozygous resistant(BB) individuals was still very steep with the proportion of BBindividuals reaching 0.99 after 19 generations. Fixation of B occurredby the 22nd generation (FIG. 4) Next, we modeled the Y allele in theabsence of both a putative or major resistance gene and any structuredrefuge. The same off-type seed rate of 0.0075 was used. Parameterizationfor the Y allele was derived from the study using selected colonies toinvestigate resistance risk in survivors of DAS-59122-7 (Lefko et al.2008). The Y allele was assumed to be relatively frequent and additivein its inheritance. Despite having a maximum YY survival rate of 0.2,the Y allele increased in frequency very rapidly (FIG. 5). Theproportion of the population responding to Y mimicked the responseobserved by Lefko et al. (2008), where the largest changes in bothcolonies selected for survival occurred after the first two generations.The proportion of XY individuals peaked at 0.6 by the 2nd generation and0.5 were YY by the 3rd generation (FIG. 5). The line predicting theproportion of individuals homozygous for Y was not as steep as the linepredicting the proportion of BB individuals when B was modeledindependently. The proportion of YY individuals was 0.90 after 7generations and 0.99 after 10 generations. Fixation of Y allele occurredby the 19th generation.

The interaction between the putative resistance gene and the tolerancetrait was assessed using the same simulation described by FIG. 3, exceptincluding the tolerance trait (Y). The proportion of each genotype overgenerations is presented in FIG. 6. Response of the tolerance traitgenotypes (XX, XY and YY) and the slope of the line predicting theproportion of individuals homozygous for Y appeared similar between thissimulation and when the tolerance trait was modeled independently (FIGS.5 and 6). The proportion of individuals with the Y allele increased veryrapidly; most of this increase came from individuals homozygoussusceptible for putative resistance (AAXY, AAYY) (FIG. 6). Theproportion of AAXY individuals peaked in the population at 0.62 afterthe 2nd generation and the proportion AAYY individuals reached 0.5 bythe 3rd generation, similar to the simulation with YY only describedabove. Individuals homozygous for Y were present at 0.9 after 7generations. An obvious sign of interaction between Y (tolerance) and B(major resistance) alleles is evident around the 9th generation when theproportion of individuals homozygous for A and Y, which had reachedapproximately 0.96, began to decline at a rate similar to its earlierrate of increase (FIG. 6).

The onset of putative resistance was significantly delayed in thissimulation incorporating both putative major resistance and thetolerance trait (FIG. 6). There were 3 genotypes heterozygous forputative resistance (ABXX, ABXY and ABYY); however, only the ABYYgenotype occurred at levels >0.01 within the population. Both the ABXXand ABXY genotypes never reached a proportion of 0.01 in the populationthroughout 30 generations of simulation. This is likely explained by thehigher initial frequency and additive inheritance of the Y allelecompared to the initial rarity and recessive inheritance of the Ballele. The rapid increase in Y allele frequency and especially AAYYindividuals is the primary explanation for the delay in resistancecompared to the simulation for the B allele alone (FIG. 6).

When resistance is recessive, the proportion of individuals heterozygousfor putative resistance (AB) generally must increase substantiallybefore resistance will evolve at an exponential rate. Any delay in theaccumulation of heterozygous (AB) individuals, prevents homozygotes andtranslates into a delay in resistance. In this simulation using arecessive putative resistance gene and a more frequent and additivetolerance trait, we observe a delay in the accumulation of ABindividuals (FIG. 6). The Y allele, and especially the ABYY genotype,interferes with the evolution of individuals heterozygous for the majorresistance allele. This is illustrated using elements of FIGS. 3, 4 and6. In the absence of both refuge and the tolerance trait (FIG. 3), theproportion of AB individuals increases rapidly until generation 5 whereBB individuals become the most abundant genotype. In the absence of thetolerance trait and presence of a 20% block refuge, the addition ofrefuge individuals (AA) results in a more gradual increase in the ABgenotype, which peaks at generation 14 (FIG. 4). The tolerance trait hasan effect on WCR adaptation toward major resistance similar to the 20%block refuge. Thus, in the absence of refuge, but presence of thetolerance trait, the proportion of ABYY genotypes increases graduallyand peak at 0.34 after generation 16 (FIG. 6). This is near generation17 when the proportion of individuals homozygous for both B and Y reach0.50. After generation 17, resistance evolves rapidly and fixation ofthe BBYY genotype occurs after 30 generations (FIG. 6). As a result, thepresence of the tolerance trait with no structured refuge hasessentially the same effect as a large block refuge in the absence of atolerance trait in delaying the development of resistant pests.

The existence of a heritable trait or traits in the rootworm population,which confer a low level of tolerance to DAS-59122-7 creates survivingrefuge insects (AA), which can extend durability relative to putativeresistance. However, the most important effect on durability stems fromthe interaction between the Y allele and putative resistance and notsimply the addition of A alleles from theses survivors. In thesesimulations, the existence of the tolerance trait increases durabilityby approximately 3-fold regardless of structured refuge beyond the0.0075 off-type rate.

This simulation shows that inheritance of a tolerance trait such as theone found in the WCR surviving DAS-59122-7 maize can significantlyprolong durability compared to a CRW system where there are no suchtolerance traits found. This is a novel finding. In this uniquesituation the Y allele confers a low level of tolerance to and survivalon DAS-59122-7. These survivors can act as refuge beetles by preservingA alleles; however, it is the interaction of a more frequent andadditive tolerance trait that is most responsible for delayingresistance. The competitiveness of this tolerance trait already in WCRpopulations introduces an adaptive barrier for development of putativeresistance. The delay in adaptation toward putative resistance based oneffects of an independent tolerance gene is similar in function to thebarrier resulting from pyramiding two insecticidal proteins withindependent mechanisms of bioactivity. In these simulations, rootwormsurviving as a direct result of a tolerance trait can extend durabilitysimilar to the durability provided by deploying a 20% block refuge inthe absence of a tolerance trait.

This simulation data, along with the empirical data collected anddescribed above, show that plants incorporating DAS-59122-7 do notrequire a separate structured refuge in order to slow the development ofresistant pests, in contrast to other events where comparable tolerancetraits are not inherent in rootworm populations. Further, the simulationdata assumes the presence of a putative resistance gene, however nopests exhibiting such a gene have yet been identified. Accordingly, thedurability of DAS-59122-7 may be even greater, for example if thedevelopment of resistance only comes about as the result of multiplegene interactions, which would most likely develop more slowly than asingle resistance allele. As a result, DAS-59122-7 is well-suited formaximizing protection against rootworms by minimizing or eliminatingstructured refuge.

Other uses for the selected colonies would be appreciated by one ofordinary skill in the art, and such uses are contemplated in the contextof the present invention. Further, while the invention has beendescribed in terms of various specific embodiments, those skilled in theart will recognize that the invention can be practiced with modificationwithin the spirit and scope of the claims.

1. A western corn rootworm exhibiting increased tolerance to eventDAS-59122-7.
 2. An egg of the rootworm of claim
 1. 3. A larva of therootworm of claim
 1. 4. A pupa of the rootworm of claim
 1. 5. A beetleof the rootworm of claim
 1. 6. The western corn rootworm of claim 1wherein the rootworm is from the York selected colony.
 7. The westerncorn rootworm of claim 1 wherein the rootworm is from the Rochelleselected colony.